Abstract:

The human genome, comprised of ~three billion base pairs, is over six feet long when fully extended. Multiple chromatin modifying enzymes are required either to condense the DNA to fit into each nucleus that are ~ 1um in size, or to partially unravel the chromatin for readout of the genomic information. The ATP-dependent chromatin remodeler INO80 plays important roles in regulating the spacing of nucleosomes along the DNA. However, mechanism by which INO80 slides the nucleosome is still unclear. Structural studies of other chromatin remodelers have been shown to be a powerful approach to understand how these enzymes interact with and modulate the positioning of nucleosome. Although X-ray crystallography can provide high resolution structure, it is not appropriate for studying INO80 because INO80 is a large protein complex (~1.3MDa) consisted of multiple subunits and modules, and only a small quantity of the protein complex can be purified. Electron microscopy (EM) is well suited for structural analysis of INO80 given the large molecular weight of the complex and the proven success of the EM approach in solving the structure of other remodelers. In this study, we used EM to study the structure and conformation range of the INO80 complex. We generated 3D class averages and 3D models of the INO80 complex based on data obtained from cryo-EM and negative stained EM. We found that INO80 is a bipartite structure with a rigid head region and an extended highly flexible tail region. The head region is a donut-shaped-structure containing the potential ATPase Rvb1/2 hexamer. On the basis of the structural features, we propose that INO80 may mediate ATP-dependent nucleosome sliding while wrapping its tail around the nucleosome.